Diffraction-based diagnostic devices

ABSTRACT

A biosensor includes a substrate with areas of active receptive material disposed thereon. The receptive material is specific for an analyte of interest. A pattern of the active areas is defined on the substrate by an oxidizing photo-masking process.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of detectinganalytes in a medium, and more particularly to a process for preparinganalyte-specific diffraction based diagnostic sensors to indicate thepresence of the analyte in a medium.

BACKGROUND

There are many systems and devices available for detecting a widevariety of analytes in various media. Many of the prior systems anddevices are, however, relatively expensive and require a trainedtechnician to perform the test. A need has been recognized in the artfor biosensor systems that are easy and inexpensive to manufacture, andcapable of reliable and sensitive detection of analytes. Reference ismade, for example, to U.S. Pat. Nos. 5,922,550; 6,060,256; and 6,221,579B1.

Various advances have been made in the industry for producingbiosensors. For example, U.S. Pat. No. 5,512,131 to Kumar, et al.,describes a device that includes a polymer substrate having a metalcoating. An analyte specific receptor layer is stamped onto the coatedsubstrate. A diffraction pattern is generated when an analyte binds tothe device. A visualization device, such as a spectrometer, is then usedto determine the presence of the diffraction pattern. A drawback to thistype of device is, however, the fact that the diffraction pattern is notdiscernible by the naked eye and, thus, a complex visualization deviceis needed to view the diffraction pattern. Also, the device is generallynot able to detect smaller analytes that do not produce a noticeablediffraction pattern.

U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device thatincludes a substrate which has an optically active surface exhibiting afirst color in response to light impinging thereon. This first color isdefined as a spectral distribution of the emanating light. The substratealso exhibits a second color which is different from the first color.The second color is exhibited in response to the same light when theanalyte is present on the surface. The change from one color to anothercan be measured either by use of an instrument, or by the naked eye. Adrawback with the device is, however, the relatively high cost of thedevice and problems associated with controlling the various layers thatare placed on the wafer substrate.

Contact printing techniques have been explored for producing biosensorshaving a self-assembling monolayer. U.S. Pat. No. 5,922,550 describes abiosensor having a metalized film upon which is printed (contactprinted) a specific predetermined pattern of an analyte-specificreceptor. The receptor materials are bound to the self-assemblingmonolayer and are specific for a particular analyte or class ofanalytes. Attachment of a target analyte that is capable of scatteringlight to select areas of the metalized plastic film upon which thereceptor is printed causes diffraction of transmitted and/or reflectedlight. A diffraction image is produced that can be easily seen with theeye or, optionally, with a sensing device. U.S. Pat. No. 6,060,256describes a similar device having a metalized film upon which is printeda specific predetermined pattern of analyte-specific receptor. The '256patent is not limited to self-assembling monolayers, but teaches thatany receptor which can be chemically coupled to a surface can be used.The invention of the '256 patent uses methods of contact printing ofpatterned monolayers utilizing derivatives of binders formicroorganisms. One example of such a derivative is a thiol. The desiredbinding agent can be thiolated antibodies or antibody fragments,proteins, nucleic acids, sugars, carbohydrates, or any otherfunctionality capable of binding an analyte. The derivatives arechemically bonded to metal surfaces such as metalized polymer films, forexample via a thiol.

A potential issue of the contact printing techniques described above forproducing diffraction-based biosensors is the possibility ofcontamination from the print surface (i.e., stamp) during the printingprocess. Also, there is the possibility of uneven application or inkingof the substances due to pressure and contact variations inherent in theprocess, as well as surface energy variations.

The present invention relates to a biosensor system that is easy andinexpensive to manufacture, is capable of reliable and sensitivedetection of analytes, and avoids possible drawbacks of conventionalmicrocontact printing techniques.

SUMMARY OF THE INVENTION

Objects and advantages of the invention will be set forth in part in thefollowing description, or may be obvious from the description, or may belearned through practice of the invention.

The present invention provides a relatively inexpensive yet sensitivebiosensor device, a method for producing such biosensor devices, and amethod for detecting and quantifying analytes of interest present in amedium.

The biosensor includes a substrate member upon which a pattern of areasof analyte specific receptive material (i.e., biomolecules) has beendefined by a negative or positive photo-oxidation masking process. Thesubstrate may be any one of a wide variety of suitable materials,including plastics, metal coated plastics and glass, functionalizedplastics and glass, silicon wafers, foils, glass, etc. Desirably, thesubstrate is flexible, such as a polymeric film, in order to facilitatethe manufacturing process.

Self assembled monolayers of thiol-containing molecules on metal,particularly gold, coated substrates form the basis for a variety ofbiosensors. Reference is made for example to U.S. Pat. No. 5,922,550,the entire disclosure of which is incorporated herein in its entiretyfor all purposes. The biosensors and masking process according to thepresent invention are based generally on the principle that, while themetal-sulfur interaction is extremely strong (nearly as strong as acovalent bond), the interaction is susceptible to oxidation, and thatthe oxidative process can be accelerated using ultraviolet (UV) light.This principle can be exploited to form a patterned monolayer ofbiomolecules on a substrate by using UV light and a patterned photomaskon a metal coated substrate to which is applied a layer of biomolecules,such as antibodies, enzymes, aptamers, or any other functionalbiomolecule.

In one particular embodiment, a generally uniform blocking monolayer ofthiol-containing molecules is first formed on a metalized substrate, forexample a gold-coated substrate (i.e., gold-coatedpolyethylene-terephthalate). The thiol-containing molecules may be, forexample, thioglucose, mercaptoethanol, or any alkanethiolate. A maskhaving any desired pattern of shielded or “protected” areas and exposedareas (transparent or translucent areas) is then placed over thesubstrate member. The mask and substrate combination are then exposed toUV light for a time sufficient to oxidize the gold-thiol link betweenthe monolayer and gold coating in the areas of the substrate exposedthrough the mask. The amount of exposure time to the UV light willdepend on the polarity and molecular size of the thiol-containingmolecules.

The substrate is then exposed (e.g., coated) with a solution of athiolated biomolecules. The substrate is exposed to the solution for asufficient length of time for a monolayer of the thiolated biomoleculesto form in the photo-oxidized areas of the substrate. The substrate isthen washed with water or a buffer to remove the excess thiolatedbiomolecules from the unoxidized areas of the substrate. The thiolatedbiomolecules remain attached to the substrate at the photo-oxidizedareas.

In a “positive” mask embodiment, the biosensor would essentially bedefined at this point with active discrete areas of receptive materialspecific for a particular analyte of interest (the thiolatedbiomolecules) defined in a pattern corresponding to the oxidized areasof the substrate. In this case, the thiolated biomolecule would bechosen such that it specifically binds a particular analyte of interest.The unoxidized areas of the substrate containing the initialthiol-containing blocking molecules define inactive non-binding areas ofthe biosensor.

In a “negative” mask embodiment, the initial thiolated biomoleculeswould be chosen such that they do not specifically bind the particularanalyte of interest. After the excess biomolecules are washed from theunoxidized areas of the substrate, the substrate is exposed again(without the mask) to the UV light source for a period of timesufficient to oxidize the remaining gold-thiol links between the initialblocking layer of thiol-containing molecules and the gold surface. Aswith the initial UV exposure, the exposure time will vary but should bemuch less than the time it would take to oxidize the gold-thiol links ofthe thiolated biomolecules attached at the first oxidized areas.

The device is then exposed to a second solution of thiolatedbiomolecules (the receptive material) selected specifically for theanalyte of interest. The exposure time is sufficient for a monolayer ofthe receptive material to form on the second oxidized areas. The excessreceptive material is then washed from the substrate. Thus, thebiosensor includes a pattern of active areas of biomolecules specificfor the analyte of interest, and a pattern of blocking or inactive areasof thiolated biomolecules that will not recognize the analyte ofinterest.

It should be appreciated that the invention is not limited to anyparticular pattern defined by the mask. Virtually any number andcombination of active shapes are possible. In one particular embodiment,the active area pattern is defined by about 10 micron diameter pixels ata spacing of about 5 microns apart over the test surface of thesubstrate.

Upon subsequent exposure of the biosensor to a medium containing ananalyte of interest, the analyte binds to the receptive material in theactive areas. The biosensor will then diffract transmitted light in adiffraction pattern corresponding to the active areas. The diffractionpattern may be visible to the naked eye or, optionally, viewed with asensing device.

In the case where an analyte does not scatter visible light because theanalyte is too small or does not have an appreciable refractive indexdifference compared to the surrounding medium, a diffraction-enhancingelement, such as polymer microparticles, may be used. Thesemicorparticles are coated with a binder or receptive material that alsospecifically binds to the analyte. Upon subsequent coupling of theanalyte to both the patterned biomolecules in the receptive materiallayer as well as the microparticles, a diffraction image is producedwhich can be easily seen with the eye or, optionally, with a sensingdevice.

By “diffraction” it is meant the phenomenon, observed when waves areobstructed by obstacles, of the disturbance spreading beyond the limitsof the geometrical shadow of the object. The effect is marked when thesize of the object is of the same order as the wavelength of the waves.In the present invention, the obstacles are analytes (with or without orattached microparticles) and the waves are light waves.

In another embodiment of the present invention, nutrients for a specificclass of microorganisms can be incorporated into the receptive materiallayer. In this way, very low concentrations of microorganisms can bedetected by first contacting the biosensor of the present invention withthe nutrients incorporated therein and then incubating the biosensorunder conditions appropriate for the growth of the bound microorganism.The microorganism is allowed to grow until there are enough organisms toform a diffraction pattern.

The present invention provides a low-cost, disposable biosensor whichcan be mass produced. The biosensors of the present invention can beproduced as a single test for detecting an analyte or it can beformatted as a multiple test device. The uses for the biosensors of thepresent invention include, but are not limited to, detection of chemicalor biological contamination in garments, such as diapers, the detectionof contamination by microorganisms in prepacked foods such as meats,fruit juices or other beverages, and the use of the biosensors of thepresent invention in health diagnostic applications such as diagnostickits for the detection of proteins, hormones, antigens, nucleic acids,microorganisms, and blood constituents. It should be appreciated thatthe present invention is not limited to any particular use orapplication.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a method for producingbiosensors according to the invention by a photo-oxidation maskingprocess.

FIG. 2 is a phase-contrast image of active anti-C-reactive proteinantibodies in a grid pattern of 10 micron squares spaced 15 micronsapart (center-to-center) in a biosensor according to the invention.

DETAILED DESCRIPTION

The invention will now be described in detail with reference toparticular embodiments thereof. The embodiments are provided by way ofexplanation of the invention, and not meant as a limitation of theinvention. For example, features described or illustrated as part of oneembodiment may be used with another embodiment to yield still a furtherembodiment. It is intended that the present invention include these andother modifications and variations as come within the scope and spiritof the invention.

The present invention features improved biosensing devices, and methodsfor using such biosensing devices, for detecting and quantifying thepresence or amount of an analyte of interest within a medium. Theanalytes that can be detected by the present invention include, but arenot limited to, microorganisms such as bacteria, yeasts, fungi,proteins, small molecules, nucleic acids, and viruses. The biosensingdevices according to the invention are relatively inexpensive and haveadvantages over conventional microcontact printed biosensors.

The present invention comprises, in broad terms, a process of definingan active pattern of analyte-specific receptor material on a substratesurface by way of a photo-oxidation masking process. The active areasmay be defined in a “negative” or “positive” masking process.

A self-assembled blocking monolayer of thiol-containing molecules isformed on a metal-coated substrate (e.g., gold-coatedpolyethylene-terephthalate). The thiol-containing molecules may be, forexample, thioglucose, mercaptoethanol, or any alkanethiolate. Althoughthere are many different systems of self-assembling monolayers based ondifferent organic components and supports, desired systems for purposesof the present invention are those of alkanethiolates, HS(CH₂)_(n)R, orthiolated saccharides (e.g., thioglucose) on gold films. Typically, agold film of 5 to 2000 nanometers thick is supported on atitanium-primed Si/Sio2 wafer or glass sheet. Alternatively, agold-coated (typically coated by plasma deposition) MYLAR®(polyethylene-terephthalate) film may be used. The alkanethiolschemisorb on the gold surface from a solution in which the gold film isimmersed, and form adsorbed alkanethiolates with loss of hydrogen.Adsorption can also occur from the vapor. The solution may also beapplied by spraying, dipping, coating, spin coating, or any othersuitable process for forming a generally uniform layer of thealkanethiolates. Self-assembling monolayers formed on gold fromlong-chain alkanethiolates of structure X(CH₂)_(n)Y(CH₂)_(m)S are highlyordered and can be considered as crystalline or quasi-crystalinemolecular arrays.

A particularly well suited metal for deposition on the substrate isgold. However, any metal that interacts with sulfur to form asulfur-metal bond is within the scope of the invention. For example,suitable metals may include silver, aluminum chromium, copper, iron,zirconium, platinum, and nickel.

Any one of a wide variety of materials may serve as the substrate towhich the receptive material and blocking material are applied. Suchmaterials are well known to those skilled in the art. For example, thesubstrate may be formed of any one of a number of suitable plastics,metal coated plastics and glass, functionalized plastics and glass,silicon wafers, glass, foils, etc. Rather than requiring a rigidsubstrate for the photopatterning process described herein, it has beenfound that thermoplastic films are quite suitable. Such films include,but are not limited to, polymers such as: polyethylene-terephthalate(MYLAR®), acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene—vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Preferably, the plasticfilm has an optical transparency of greater than 80 percent. Othersuitable thermoplastics and suppliers may be found, for example, inreference works such as the Modern Plastics Encyclopedia (McGraw-HillPublishing Co., New York 1923-1996).

The film with metal coating thereon may have an optical transparency ofbetween approximately 5 percent and 95 percent. A more desired opticaltransparency for the thermoplastic film used in the present invention isbetween approximately 20 percent and 80 percent. In a desired embodimentof the present invention, the thermoplastic film has at least anapproximately 80 percent optical transparency, and the thickness of themetal coating is such as to maintain an optical transparency greaterthan about 20 percent, so that diffraction patterns can be produced byeither reflected or transmitted light. This corresponds to a metalcoating thickness of about 20 nanometers. However, in other embodimentsof the invention, the metal thickness may be between approximately 1nanometer and 1000 nanometers.

A mask having any desired pattern of exposed and protected regions isthen placed over the substrate and the mask and substrate combination isirradiated with UV light. The mask may be formed of any suitablematerial that protects or blocks portions of the underlying substratefrom the UV light source. A material that has proven useful in thephoto-oxidation masking process on a gold-coated MYLAR® film coated withthe blocking layer is a transparent or translucent polymer film (such asMYLAR®) having a pattern of shielded or protected regions printedthereon. This type of mask is useful for light sources with a wavelengthequal to or greater than about 330 nanometers. For light sources havinga wavelength below about 330 nanometers, a quartz or fused silica maskhaving chrome plated shielded regions defined thereon may be used. Themask may define any pattern of oxidized and unoxidized regions of theblocking layer. The respective regions creating the visible diffractionpattern may be of virtually any size, shape, and pattern. It may bedesired to select a size and pattern so as to maximize the visiblediffraction contrast between the active and inactive regions. As oneexample, it has been found suitable if the active regions are defined asgenerally circular with a diameter of about 10 microns and spaced fromeach other by about 5 microns.

The time of exposure to the UV light required for oxidizing the exposedareas of the blocking layer will depend on the size and polarity of thethiol-containing molecules. “Small” molecules less than about 500molecular weight (MW) and more polar molecules are desired in that theoxidizing reaction is more rapid with such molecules. The UV lightcauses rapid oxidation of the sulfur in the thiol-containing blockinglayer and, thus, degradation of the metal-thiol bond at the oxidizedsites.

After exposure to the UV light and removal of the mask, a pattern ofoxidized areas is defined. These areas will become either active areasof receptive material (positive masking) or shielded inactive areas(negative masking).

The substrate is then exposed to a solution of a thiolated biomolecule.The solution may be applied by any appropriate method, such as dipping,spraying, coating, spin coating, etc. The solution essentially washesaway the oxidized molecules (sulfates) such that a bare metal surface isexposed at the oxidized sites. It should be appreciated that anintervening washing step may be carried out for this purpose prior toexposing the substrate to the thiolated biomolecule. The exposure timeis sufficient for a monolayer of the thiolated biomolecules to form onthe substrate at the oxidized sites. It should be ensured that theconcentration of the biomolecule agent in the solution is sufficient todeposit a relatively uniform layer of the material on the substrate. Ina “positive” masking process, the biomolecule is selected to be specificfor a particular analyte of interest. In other words, the biomoleculesrecognize the target ligand. In a “negative” masking process, thebiomolecule is a blocking agent and is selected specifically not torecognize the analyte of interest.

The thiolated biomolecules thus displace the oxidized blocking moleculesand attach to the metal surface with a strong thiol-metal bond. Theexcess thiolated biomolecules are disassociated from the unoxidizedareas of the substrate by washing or rinsing the substrate with water ora buffer solution.

For a positive masking process, the thiolated biomoleclues attached atthe ozxidized regions may be specific for the analyte of interest. Inthis case, localized active areas of receptive material are defined in apattern corresponding to the oxidized areas and the remaining regions ofthe substrate with the initial blocking layer define shielded orinactive areas.

For a negative masking process, the substrate is exposed again (withoutthe mask) to the UV light source for a period of time sufficient tooxidize the remaining metal-thiol links between the initial blockinglayer of thiol-containing molecules and the metal surface (the secondoxidized areas) without oxidizing the metal-thiol links of the thiolatedbiomolecules attached at the first oxidized areas. The substrate is thenexposed to a second solution of thiolated biomolecules selectedspecifically for the analyte of interest by dipping, rolling, spraying,coating, etc. The solution washes away the oxidized molecules (sulfates)from the second oxidized areas and a monolayer of the second thiolatedbiomolecules is formed on the substrate at the second oxidized areas,with the biomolecules specifically attaching to the metal surface at thesecond oxidized areas by a metal-thiol bond. The excess receptivematerial is then washed from the substrate. Thus, the biosensor includesa pattern of active areas of biomolecules specific for the analyte ofinterest, and a pattern of blocking or inactive areas of thiolatedbiomolecules that will not recognize the analyte of interest.

It should be understood that “pattern” includes as few as one activearea and one inactive area.

Upon subsequent exposure of the biosensor to a medium containing theanalyte of interest, such analyte will bind to the receptors in theactive receptive material areas. The analyte results in diffraction oftransmitted and/or reflected light in a visible diffraction patterncorresponding to the active areas. As discussed in greater detail below,an enhancer may be used for enhancing diffraction from extremely smallanalytes.

The analytes that are contemplated as being detected using the presentinvention include, but are not limited to, bacteria; yeasts; fungi;viruses; rheumatoid factor; antibodies, including, but not limited toIgG, IgM, IgA, IgD, and IgE antibodies; carcinoembryonic antigen;streptococcus Group A antigen; viral antigens; antigens associated withautoimmune disease; allergens; tumor antigens; streptococcus Group Bantigen; HIV I or HIV II antigen; or host response (antibodies) to theseand other viruses; antigens specific to RSV or host response(antibodies) to-the virus; antigen; enzyme; hormone; polysaccharide;protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species;Candida species, including, but not limited to Candida albicans andCandida tropicalis; Neisseria meningitides groups A, B, C, Y and W sub135, Streptococcus pneumoniae, E. coli, Haemophilus influenza type A/B;an antigen derived from microorganisms; PSA and CRP antigens; a hapten;a drug of abuse; a therapeutic drug; an environmental agent; andantigens specific to Hepatitis. In broad terms, the “analyte ofinterest” may be thought of as any agent whose presence or absence froma biological sample is indicative of a particular health state orcondition.

It is also contemplated that nutrients for a specific class ofmicroorganism can be incorporated into the receptive material layer. Inthis way, very low concentrations of microorganisms can be detected byexposing the biosensor of the present invention with the nutrientsincorporated therein to the suspect medium and then incubating thebiosensor under conditions appropriate for the growth of the boundmicroorganism. The microorganisms are allowed to grow until there areenough organisms to form a diffraction pattern. Of course, in somecases, the microorganism is present or can multiply enough to form adiffraction pattern without the presence of a nutrient in the activereceptive material areas.

The receptive material is characterized by an ability to specificallybind the analyte or analytes of interest. The variety of materials thatcan be used as receptive material is limited only by the types ofmaterial which will combine selectively (with respect to any chosensample) with a secondary partner. Subclasses of materials which fall inthe overall class of receptive materials include toxins, antibodies,antibody fragments, antigens, hormone receptors, parasites, cells,haptens, metabolites, allergens, nucleic acids, nuclear materials,autoantibodies, blood proteins, cellular debris, enzymes, tissueproteins, enzyme substrates, coenzymes, neuron transmitters, viruses,viral particles, microorganisms, proteins, polysaccharides, chelators,drugs, aptamers, peptides and any other member of a specific bindingpair. This list only incorporates some of the many different materialsthat can be coated onto the substrate surface to produce a thin filmassay system. Whatever the selected analyte of interest is, thereceptive material is designed to bind specifically with the analyte ofinterest.

The matrix or medium containing the analyte of interest may be a liquid,a solid, or a gas, and can include a bodily fluid such as mucous,saliva, urine, fecal material, tissue, marrow, cerebral spinal fluid,serum, plasma, whole blood, sputum, buffered solutions, extractedsolutions, semen, vaginal secretions, pericardial, gastric, peritoneal,pleural, or other washes and the like. The analyte of interest may be anantigen, an antibody, an enzyme, a DNA fragment, an intact gene, a RNAfragment, a small molecule, a metal, a toxin, an environmental agent, anucleic acid, a cytoplasm component, pili or flagella component,protein, polysaccharide, drug, or any other material. For example,receptive material for bacteria may specifically bind a surface membranecomponent, protein or lipid, a polysaccharide, a nucleic acid, or anenzyme. The analyte which is specific to the bacteria may be apolysaccharide, an enzyme, a nucleic acid, a membrane component, or anantibody produced by the host in response to the bacteria. The presenceor absence of the analyte may indicate an infectious disease (bacterialor viral), cancer or other metabolic disorder or condition. The presenceor absence of the analyte may be an indication of food poisoning orother toxic exposure. The analyte may indicate drug abuse or may monitorlevels of therapeutic agents.

One of the most commonly encountered assay protocols for which thistechnology can be utilized is an immunoassay. However, the generalconsiderations apply to nucleic acid probes, enzyme/substrate, and otherligand/receptor assay formats. For immunoassays, an antibody may serveas the receptive material or it may be the analyte of interest. Thereceptive material, for example an antibody or an antigen, should form astable, reactive layer on the substrate surface of the test device. Ifan antigen is to be detected and an antibody is the receptive material,the antibody must be specific to the antigen of interest; and theantibody (receptive material) must bind the antigen (analyte) withsufficient avidity that the antigen is retained at the test surface. Insome cases, the analyte may not simply bind the receptive material, butmay cause a detectable modification of the receptive material to occur.This interaction could cause an increase in mass at the test surface, adecrease in the amount of receptive material on the test surface, or achange in refractive index. An example of the latter is the interactionof a degradative enzyme or material with a specific, immobilizedsubstrate. In this case, one would see a diffraction pattern beforeinteraction with the analyte of interest, but the diffraction patternwould disappear if the analyte were present. The specific mechanismthrough which binding, hybridization, or interaction of the analyte withthe receptive material occurs is not important to this invention, butmay impact the reaction conditions used in the final assay protocol.

In addition to producing a simple diffraction image, patterns ofanalytes can be such as to allow for the development of a holographicsensing image and/or a change in visible color. Thus, the appearance ofa hologram or a change in an existing hologram will indicate a positiveresponse. The pattern made by the diffraction of the transmitted lightcan be any shape including, but not limited to, the transformation of apattern from one pattern to another upon binding of the analyte to thereceptive material. In particularly preferred embodiments, thediffraction pattern becomes discernible in less than one hour aftercontact of the analyte with the biosensing device of the presentinvention.

The diffraction grating which produces the diffraction of light uponinteraction with the analyte must have a minimum periodicity of about ½the wavelength and a refractive index different from that of thesurrounding medium. Very small analytes, such as viruses or molecules,can be detected indirectly by using a larger, “diffraction-enhancingelement,” such as a micro-particle, that is specific for the smallanalyte. One embodiment in which the small analyte can be detectedcomprises coating the enhancing particle, such as a latex bead orpolystyrene bead, with a receptive material, such as an antibody, thatspecifically binds to the analyte of interest. Particles that can beused in the present invention include, but are not limited to, glass,cellulose, synthetic polymers or plastics, latex, polystyrene,polycarbonate, proteins, bacterial or fungal cells, silica, celluloseacetate, carbon, and the like. The particles are desirably spherical inshape, but the structural and spatial configuration of the particles isnot critical to the present invention. For instance, the particles couldbe slivers, ellipsoids, cubes, random shape and the like. A desirableparticle size ranges from a diameter of approximately 0.1 micron to 50microns, desirably between approximately 0.1 micron and 2.0 microns. Thecomposition of the particle is not critical to the present invention.

Desirably, the receptive material layer on the substrate willspecifically bind to an epitope on the analyte that is different fromthe epitope used in the binding to the enhancing particle. Thus, fordetecting a small analyte in a medium, the medium is first exposed tothe latex particles having the virus-specific receptive materialthereon. The small analytes of interest in the medium will bind to thelatex particles. Then, the latex particles are optionally washed andexposed to the biosensor film with the pattern of active receptivematerial areas containing the virus-specific antibodies. The antibodiesthen bind to the viral particles on the latex bead thereby immobilizingthe latex beads in the same pattern as the active areas on the film.Because the bound latex beads will cause diffraction of the visiblelight, a diffraction pattern is formed, indicating the presence of theviral particle in the liquid. Other combinations using diffractionenhancing particles are described, for example, in U.S. Pat. No.6,221,579 incorporated herein for all purposes.

The first and second thiolated biomolecule solutions may be applied tothe substrate by any conventional method. The material is applied sothat it generally uniformly covers an entire (for example, upper)surface of the substrate. Non-contact methods for applying the receptivematerial may be desired so as to eliminate the possibility ofcontamination by contact during application. Suitable applicationmethods include, but are not limited to, dipping, spraying, rolling,spin coating, and any other technique wherein the solutions can beapplied generally uniformly over the entire test surface of thesubstrate.

The technique selected should minimize the amount of biomoleculesolution required for coating a large number of test surfaces andmaintain the stability/functionality of the receptive material duringapplication. The technique should also apply or adhere the receptivematerial to the substrate in a uniform and reproducible fashion.

FIG. 1 is a schematic representation of one method for producingbiosensors by a photo-oxidation method according to the invention. StepA represents a thiol-containing blocking layer 2 applied to a substratemember 4. Step B represents a mask 6 disposed over the substrate member4. The mask 6 includes exposed or open regions 8 and shielded orprotected regions 10 defined thereon. The mask 6 and substrate 4combination are exposed to a UV light 12. The areas of the substrate 4under the protected regions 10 of the mask 6 are protected from thelight 12, and the areas of the blocking layer 2 under the open areas 8of the mask 6 are exposed to the light 12. Step C represents the deviceafter exposure to the light 12 and removal of the mask 6. The blockinglayer material 2′ exposed to the light 12 has been oxidized such that apattern of oxidized areas are defined on the substrate member 4. Step Drepresents the device after it has been exposed to a first thiolatedbiomolecule solution. The oxidized particles 2′ have been displaced by amonolayer of the thiolated biomolecules 14. For a positive maskingprocedure, the biomolecules 14 are specific for the analyte of interestand the biosensor is essentially defined at this point. For a negativemasking procedure, the biomolecules 14 are a blocking agent and thefurther steps are taken to define the active areas of receptivematerial. Step E-represents the device being exposed a second time tothe UV light 12 without the mask 6. The exposure time is sufficient tooxidize the remaining areas of the initial blocking layer material 2without disrupting the gold-thiol bond between the substrate member 4and biomolecules 14. Step F represents the device after the secondexposure. The remaining blocking layer material 2 is oxidized(represented by 2″) and the areas of biomolecules 14 remain intact. StepG represents the device after it has been exposed to a second solutionof thiolated biomolecules. The oxidized particles 2″ have been displacedby the second thiolated biomolecules 16 that are specific for theanalyte of interest. At this point, the biosensor is defined by apattern of active receptive material areas (areas of biomolecule 16) anda pattern of inactive or shielded areas (areas of blocking biomolecule14).

The biosensors according to the invention have a wide range of uses inany number of fields. The uses for the biosensors of the presentinvention include, but are not limited to, detection of chemical orbiological contamination in garments, such as diapers, generally thedetection of contamination by microorganisms in prepacked foods such asmeats, fruit juices or other beverages, and the use of the biosensors ofthe present invention in health diagnostic applications such asdiagnostic kits for the detection of proteins, hormones, antigens, DNA,microorganisms, and blood constituents. The present invention can alsobe used on contact lenses, eyeglasses, window panes, pharmaceuticalvials, solvent containers, water bottles, band-aids, wipes, and the liketo detect contamination. In one embodiment, the present invention iscontemplated in a dipstick form in which the patterned substrate ismounted at the end of the dipstick. In use the dipstick is dipped intothe liquid in which the suspected analyte may be present and allowed toremain for several minutes. The dipstick is then removed and then,either a light is projected through the substrate or the substrate isobserved with a light reflected from the substrate. If a diffractionpattern is observed, then the analyte is present in the liquid.

In another embodiment of the present invention, a multiple analyte testis constructed on the same support. A strip may be provided with severalpatterned substrate sections. Each section has a different receptivematerial that is different for different analytes. It can be seen thatthe present invention can be formatted in any array with a variety ofpatterned substrates thereby allowing the user of the biosensor deviceof the present invention to detect the presence of multiple analytes ina medium using a single test.

In yet another embodiment of the present invention, the biosensor can beattached to an adhesively backed sticker or decal which can then beplaced on a hard surface or container wall. The biosensor can be placedon the inside surface of a container such as a food package or a glassvial. The biosensor can then be visualized to determine whether there ismicrobial contamination.

The invention is further illustrated by the following example, which arenot to be construed in any way as imposing limitations upon the scope ofthe invention. It should be understood that resort may be had to variousother embodiments, modifications, and equivalents thereof, which, afterreading the description of the invention herein, may suggest themselvesto those skilled in the art without departing from the scope and spiritof the present invention.

EXAMPLE 1

Gold-coated mylar (gold thickness˜10 nanometer, <20 ohms resistance,total thickness of 3-7 mils, from Courtaulds Performance Films, CanogaPark, Calif.) was exposed to a 100 mM solution of thio-b-1-D-glucose for5 min followed by a rinse with water. The film was blown dry and thenmounted in a vacuum frame in intimate contact with a photomask. Thechrome-on-quartz photomask was produced by direct-write e-beam with apattern that was a regular grid of 10 um diameter squares spaced 15 umcenter-to-center (positive image). The film was exposed to broadband UVlight using a solar simulator without filters (LS-1000, Solar Light,Philadelphia, Pa.) for 10 min. The film was removed from the vacuumframe and exposed for 10 min to a 1 mg/ml solution of thiolatedanti-lutenizing hormone beta subunit (LH) monoclonal antibody(Fitzgerald, #10-115, lot#191) The antibody was thiolated usingSulfo-LC-SPDP (Pierce cat # 21650ZZ) and purified using gel-filtration.After exposure to the anti-LH antibody the film was exposed for anadditional 10 min to broadband UV light using the solar simulator,however without a photomask. The film was then dipped in a 1 mg/mlsolution of thiolated anti-C-reactive protein (CRP) monoclonal antibody(Biospacific, #A58040136P, lot# A0640) for 10 min. The anti-CRP antibodywas thiolated in the same manner as the anti-LH antibody. After exposureto the anti-CRP antibody, the film was washed with filtered water andblown dry with filtered air.

The resulting pattern of anti-CRP antibody zones was visualized using anenzyme-based assay that generates a colored precipitate. A 1 ug/mLsolution of C-reactive protein that was covalently linked to horseradishperoxidase (Dako, #P0227, lot#074-301) was reacted with the patternedantibody surface for 10 min followed by a rinse with PBS (50 mM, pH 7.4phosphate buffer, 150 mM sodium chloride). The slide was then blown drywith filtered air. The residual horseradish peroxidase (localized to theactive zones via antibody recognition of the C-reactive protein) wasvisualized by precipitation of tetramethyl benzidine (KPL Microwellperoxidase substrate, #50-76-04 and KPL Membrane Enhancer, #50-77-01).

1. A method for producing biosensors, comprising: forming a blockinglayer of thiol-containing molecules on a metalized substrate member, thethiol-containing molecules forming thiol-metal bonds with the metalsurface of the substrate member; exposing the substrate member to anoxidizing enhancing stimulus through a mask having a pattern of exposedareas and protected areas, said exposure being of sufficient time tooxidize the thiol-metal bonds in the exposed areas of the blocking layerunder the mask; exposing the substrate member to a first source ofthiolated biomolecules without an intervening washing step, for a timesufficient to remove the oxidized thiol-metal bonds in the exposed areasand form a layer of the first thiolated biomolecules directly on themetalized substrate member at the oxidized areas of the blocking layerby displacing the oxidized thiol-containing molecules in the exposedareas with the first thiolated biomolecule, the first thiolatedbiomolecules being specific for an analyte of interest having aparticular size, the first thiolated biomolecules defining active areasof receptive material in a pattern corresponding to the oxidized areasdefined by the exposing to oxidize step, wherein when the biosensor isexposed to a medium containing the analyte of interest, the analytebinds to the receptive material in the active areas such that theparticular size of the analyte of interest, with or without adiffraction enhancing element, facilitates subsequent diffraction oflight in a diffraction pattern corresponding to the active areas.
 2. Themethod as in claim 1, wherein the first thiolated biomolecules areselected from at least one of antigens, antibodies, nucleotides,chelators, enzymes, bacteria, yeasts, fungi, viruses, bacterial pili,bacterial flagellar materials, nucleic acids, polysaccharides, lipids,proteins, carbohydrates, metals, hormones, and respective receptors forsaid materials.
 3. The method as in claim 1, wherein the oxidizingenhancing stimulus is a UV light source.
 4. The method as in claim 1,wherein the metalized substrate is gold-coated.
 5. The method as inclaim 1, comprising selecting the substrate member from the group ofmaterials consisting of plastics, metal coated plastics and glass,functionalized plastics and glass, silicon wafers, and foils.
 6. Themethod as in claim 1, wherein the substrate member comprises a polymerfilm coated with a metal.
 7. The method as in claim 6, wherein thepolymer film comprises polyethylene-terephthalate.
 8. The method as inclaim 6, comprising selecting the metal from the group consisting ofgold, silver, chromium, nickel, platinum, aluminum, iron, copper, goldoxide, chromium oxide and zirconium.
 9. The method as in claim 1,comprising viewing a diffraction pattern of active areas with a nakedeye.
 10. The method as in claim 1, wherein the analyte of interest isselected from at least one of a bacteria, yeast, fungus, virus,rheumatoid factor, IgG, IgM, IgA, IgD and IgE antibodies,carcinoembryonic antigen, streptococcus Group A antigen, viral antigens,antigens associated with autoimmune disease, allergens, tumor antigens,streptococcus group B antigen, IIV I or HIV II antigen, antibodiesviruses, antigens specific to RSV, an antibody, antigen, enzyme,hormone, polysaccharide, protein, lipid, carbohydrate, drug, nucleicacid, Neisseria meningitides groups A, B, C, Y and W sub 135,Streptococcus pneumoniae, E. coli K1, Haemophllus influenza type A/B, anantigen derived from microorganisms, PSA and CRP antigens, a hapten, adrug of abuse, a therapeutic drug, an environmental agents, or antigensspecific to Hepatitis.
 11. The method as in claim 1, wherein the firstthiolated biomolecules are non-specific for a particular analyte ofinterest, and further comprising exposing the substrate member a secondtime to the oxidizing stimulus without the mask for a sufficient time tooxidize the thiol-metal bonds in the remaining areas of the blockinglayer, and subsequently exposing the substrate member to a second sourceof thiolated biomolecules that are specific for the analyte of interestfor a time sufficient for a layer of the second thiolated biomoleculesto form at the second oxidized areas, the areas containing the secondthiolated biomolecules defining active areas of receptive material, andthe areas containing the first thiolated biomolecules defining shieldedareas of the substrate, and wherein when the biosensor is exposed to amedium containing the analyte of interest, the analyte binds to thereceptive material in the active areas and facilitate subsequentdiffraction of transmitted light in a diffraction pattern correspondingto the active areas.
 12. The method as in claim 11, wherein the secondthiolated biomolecules are selected from at least one of antigens,antibodies, nucleotides, chelators, enzymes, bacteria, yeasts, fungi,viruses, bacterial pili, bacterial flagellar materials, nucleic acids,polysaccharides, lipids, proteins, carbohydrates, metals, hormones, andrespective receptors for said materials.
 13. The method as in claim 11,wherein the analyte of interest is selected from at least one of abacteria, yeast, fungus, virus, rheumatoid factor, IgG, IgM, IgA, IgDand IgE antibodies, carcinoembryonic antigen, streptococcus Group Aantigen, viral antigens, antigens associated with autoimmune disease,allergens, tumor antigens, streptococcus group B antigen, IIV I or HIVII antigen, antibodies viruses, antigens specific to RSV, an antibody,antigen, enzyme, hormone, polysaccharide, protein, lipid, carbohydrate,drug, nucleic acid, Neisseria meningitides groups A, B, C, Y and W sub135, Streptococcus pneumoniae, E. coil K1, Haemophllus influenza typeA/B, an antigen derived from microorganisms, PSA and CRP antigens, ahapten, a drug of abuse, a therapeutic drug, an environmental agents, orantigens specific to Hepatitis.
 14. The method as in claim 11, whereinthe oxidizing enhancing stimulus is a UV light source.
 15. The method asin claim 11, wherein the metalized substrate is gold-coated.
 16. Themethod as in claim 11, comprising selecting the substrate member fromthe group of materials consisting of plastics, metal coated plastics andglass, functionalized plastics and glass, silicon wafers, and foils. 17.The method as in claim 11, wherein the substrate member comprises apolymer film coated with a metal.
 18. The method as in claim 17, whereinthe polymer film comprises polyethylene-terephthalate.
 19. The method asin claim 1, wherein the thiol-containing molecules consist essentiallyof thioglucose.